LncRNA HOTAIR impairs the prognosis of papillary thyroid cancer via regulating cellular malignancy and epigenetically suppressing DLX1

Purpose Papillary thyroid cancer (PTC) is the most common endocrine malignancy with a fast-growing incidence in recent decades. HOTAIR as a long non-coding RNA has been shown to be highly expressed in papillary thyroid cancer tissues with only a limited understanding of its functional roles and downstream regulatory mechanisms in papillary thyroid cancer cells. Methods We applied three thyroid cancer cell lines (MDA-T32, MDA-T41 and K1) to investigate the phenotypic influence after gain or loss of HOTAIR. The Cancer Genome Atlas (TCGA) database were utilised to select candidate genes possibly regulated by HOTAIR with validation in the cellular system and immunohistochemical (IHC) staining of PTC tissues. Results We observed HOTAIR was highly expressed in MDA-T32 cells but presents significantly decreased levels in MDA-T41 and K1 cells. HOTAIR knockdown in MDA-T32 cells significantly suppressed proliferation, colony formation, migration with cell cycle retardation at G1 phase. On the contrary, HOTAIR overexpression in MDA-T41 cells dramatically enhanced proliferation, colony formation, migration with cell cycle driven toward S and G2/M phases. Similar phenotypic effects were also observed as overexpressing HOTAIR in K1 cells. To explore novel HOTAIR downstream mechanisms, we analyzed TCGA transcriptome in PTC tissues and found DLX1 negatively correlated to HOTAIR, and its lower expression associated with reduced progression free survival. We further validated DLX1 gene was epigenetically suppressed by HOTAIR via performing chromatin immunoprecipitation. Moreover, IHC staining shows a significantly stepwise decrease of DLX1 protein from normal thyroid tissues to stage III PTC tissues. Conclusions Our study pointed out that HOTAIR is a key regulator of cellular malignancy and its epigenetic suppression on DLX1 serves as a novel biomarker to evaluate the PTC disease progression. Supplementary Information The online version contains supplementary material available at 10.1186/s12935-022-02817-2.


Introduction
Thyroid cancer is the most common endocrine malignancy and presents a rapid growing annual incidence in recent decades. The increased incidence is probably due to early detection using high-resolution ultrasonography and commonly diagnosed at a younger age [1].
Histologically, there are five major types of thyroid cancer including papillary, follicular, poorly differentiated, anaplastic, and medullary thyroid cancer. Among them, papillary thyroid cancer (PTC) accounts for the majority (around 80%) of thyroid cancer [2]. The first-line treatment of PTC is total thyroidectomy, then followed with radioactive iodine ablation of thyroid remnant and thyroid hormone suppression of thyroid-stimulating hormone (TSH). Generally, the overall survival is more than 90% [3]. However, recent research disclosed there are still around 20% of PTC patients encountered disease recurrence with lymph nodes metastasis, causing the consequence of increased mortality [4]. Indeed, heterogeneity within the category of PTC has been reported and only could be roughly characterized by some histological features and molecular markers, such as BRAF V600E mutation and RET/PTC rearrangements [5]. Besides, using transcriptional and mutational landscape, welldifferentiated thyroid cancer could be classified as three molecular subtypes including BRAF-like, RAS-like, and Non-BRAF-Non-RAS [6]. Notably, increased prevalence of metastasis, gross extra-thyroidal extension, and multifocality were found in the BRAF-like subtype [7]. Therefore, further investigation on the genetic and epigenetic alternations of BRAF-like PTC to identify novel prognosis associated markers is of vital importance.
Long non-coding RNAs (lncRNAs) were defined as RNA transcripts with more than 200 nucleotides and not translated into proteins. Recently, many lncRNAs have been recognized to present dynamic expression during the mammalian organ development [8] and their dysregulations were observed among numerous cancers [9], indicating lncRNAs could function as important regulators during organogenesis and involve in carcinogenesis. HOTAIR (HOX Transcript Antisense Intergenic RNA), as one of the well-known lncRNAs, has recently been noted to possess potential roles in PTC. HOTAIR could be detected in serum and the levels might serve as a diagnostic value to distinguish thyroid benign nodule and PTC [10]. Also, PTC patients with lymph node metastasis have significantly higher serum HOTAIR levels than them without metastasis [11]. Moreover, PTC tissues were shown to express increased HOTAIR levels, which further positively correlated with advanced pathological stages and poor prognosis of patients [12]. These results point out HOTAIR likely plays functional roles during PTC carcinogenesis and metastasis and can serve as a potential marker for evaluating disease status of PTC patients.
To explore HOTAIR mediated molecular mechanisms in thyroid cancer, several groups have examined the HOTAIR-miRNA-mRNA competitive endogenous RNA networks in multiple thyroid cancer cell lines. For examples, using papillary (TPC-1) and follicular (FTC-133) thyroid cancer cell lines, HOTAIR was showed to sponge miR-1, activate CCND2 expression, and promote thyroid cancer progression [13]. Also, HOTAIR can down-regulate miR-488-5p with upregulation of NUP205 and Bcl-2 to enhance growth, migration, and invasion of the papillary (BCPAP) thyroid cancer cell [14]. Similarly, HOTAIR promotes cell viability, migration, and invasion in papillary (TPC-1) and follicular (FTC-133) thyroid cancer cells via counter-regulating miR-17-5p [15]. Using papillary (BCPAP) and anaplastic (HTh-7 and CAL-62) thyroid cancer cells, HOTAIR/miR-761 sponge can regulate PPME1 to promote cell proliferation and inhibit cell apoptosis [16]. Therefore, it is undoubtedly that HOTAIR can sponge various miRNAs to enhance cell malignant behaviours in thyroid cancer cells.
However, HOTAIR mediated epigenetic mechanisms are not limited to miRNAs sponge. HOTAIR can also silence genes in trans via interacting with polycomb repressive complex 2 (PRC2) at the 5' end and lysine-specific histone demethylase 1 (LSD1) at the 3' end to modulate H3K27 trimethylation and H3K4 demethylation, respectively [17]. To our knowledge, this mechanism in papillary thyroid cancer has not been investigated before. Additionally, previous research mainly used the traditional PTC cell lines, such as BCPAP and TPC-1. Therefore, in this study, we utilized two newly characterized PTC cell lines (MDA-T32 and MDA-T41) [18] to evaluate the HOTAIR phenotypic effects on cellular biology after gain or loss of its expression. The Cancer Genome Atlas (TCGA) database were used to select genes that potentially repressed by HOTAIR in PTC tissues. The cellular models, chromatin immunoprecipitation assay and immunohistochemical (IHC) staining of PTC tissues were further applied to validate the putative HOTAIR-suppressed gene and evaluate its association with the clinical staging of PTC. We aim to strengthen current understandings of HOTAIR-mediated carcinogenesis and epigenetic alternations in PTC with identification of novel prognosis associated markers for clinical application.

HOTAIR was overexpressed in PTC, associated with decreased overall survival, and variably expressed among four PTC cell lines
Using the TCGA database, we compare the gene expression between PTC tissues and their matched adjacent normal thyroid tissues (n = 59) and the PTC tissues were shown to present significantly increased HOTAIR levels (Fig. 1A). Besides, the elevated HOTAIR expression was further associated with reduced survival probability in PTC patients (Fig. 1B), suggesting HOTAIR might play functional roles to steer the tumorigenesis and disease progression of PTC. Previous studies have found the BCPAP cells express significantly higher HOTAIR levels than the normal thyroid follicular cells [14,15]. Here, we further compared HOTAIR expression among four PTC cells (BCPAP, K1, MDA-T41 and MDA-T32). Notably, although these PTC cell lines all carried BRAF V600E mutation, but they expressed very differential HOTAIR levels (Fig. 1C), implying HOTAIR might be a BRAF V600E mutation independent epigenetic regulator to alter the malignant behaviours of PTC cells. Using the TCGA database, the HOTAIR levels among the PTCs with different genetic background including BRAF V600E , NRAS, HRAS mutations and RET fusion were further compared (Additional file 1: Figure S1). Although the results showed no significant difference, but the PTCs with BRAF V600E mutation do have high individual variation on HOTAIR expression, which is in line with our findings that there are differential HOTAIR levels among four PTC cell lines with BRAF V600E mutation. HOTAIR was variably overexpressed in PTC tissues and cell lines and associated with survival probability. A HOTAIR expression in PTC tissues and corresponding adjacent normal thyroid tissues were obtained from TCGA database and specifically compared. B Kaplan-Meier survival analysis revealed patients with high HOTAIR expression (n = 96) have decreased overall survival comparing to ones with low HOTAIR levels (n = 417). Cut off value: 0.4078 C Relative HOTAIR expression was measured by real-time qPCR and compared between four PTC cell lines (n = 3). Data are presented as means ± SD. Wilcoxon Signed Ranks test was used to compare HOTAIR levels between PTC tissues and adjacent normal thyroid tissues. Independent Student T-test for comparing HOTAIR levels among PTC cell lines. Log-rank test was used to compare survival probability. *p < 0.05; **p < 0.01; ***p < 0.001

Modulation of HOTAIR expression in two well-characterized PTC cells (MDA-T41 and MDA-T32) regulates cell proliferation, colony formation and cell migration
Since MDA-T32 and MDA-T41 are the newly developed and well-characterized PTC cells [18] with differential HOTAIR expression (MDA-T32 presents highest HOTAIR expression with 6.4-fold higher levels than that in MDA-T41) (Fig. 1C), we applied MDA-T32 and MDA-T41 cell lines to perform HOTAIR knockdown and constitutive overexpression experiments, respectively. As shown in Fig. 2A and E, we successfully knocked down HOTAIR transcript in MDA-T32 and induced HOTAIR overexpression in MDA-T41. Then, HOTAIR knockdown in MDA-T32 substantially suppressed the cell proliferation rate (Fig. 2B), colony formation (Fig. 2C) and cell migration (Fig. 2D). In contrast, constitutive overexpression of HOTAIR in MDA-T41 remarkably increased the cell proliferation rate (Fig. 2F), colony formation (Fig. 2G) and cell migration (Fig. 2H). These results provide supportive evidence that HOTAIR as a lncRNA plays active roles to promote the cell malignant behaviours in PTC.  3). B, C Cell proliferation rate of Scramble and si-HOTAIR cells was assessed by CCK-8 and colony formation assay (n = 3). D Cell migration of Scramble and si-HOTAIR cells was evaluated using wound-healing assay (n = 3). E Constitutive HOTAIR overexpression in the MDA-T41 cell line (HOTAIR-OE cell) was generated via lentiviral transduction and empty vector was utilized to generate the Control cell (n = 3). F, G Cell proliferation rate of Control and HOTAIR-OE cells was assessed by CCK-8 and colony formation assay (n = 3). H Cell migration of Control and HOTAIR-OE cells was evaluated using wound-healing assay (n = 3). Data are presented as means ± SD. Statistical significance was assessed using independent Student T-test. *p < 0.05; **p < 0.01; ***p < 0.001

Modulation of HOTAIR in PTC cell lines alters cell cycle phases and PTEN/p-AKT pathway
HOTAIR influences the cell proliferation rate and colony formation in PTC cell lines might base on its effects on cell cycle phases. Therefore, we further examine its effects on cell cycle phases via running flow cytometry and blotting the corresponding proteins. Significantly, HOTAIR knockdown in MDA-T32 cells retarded the cell cycle in the G1 phase with decreased ratio of G2/M phase distribution (Fig. 3A). Compatibly, in the MDA-T32 si-HOTAIR cells, G1 phase related proteins (CDK6 and Cyclin D1) were significantly increased, while S phase related proteins (CDK2 and Cyclin E1) and G2/M phase related proteins (CDK1, Cyclin A2 and Cyclin B1) were inhibited (Fig. 3B). On the contrary, HOTAIR overexpression in MDA-T41 cells shifted the cell cycle toward S and G2/M phases with decreased ratio of G1 phase distribution (Fig. 3C). Also, in the MDA-T41 HOTAIR-OE cells, S phase related proteins (CDK2 and Cyclin E1) and G2/M phase related proteins (CDK1, Cyclin A2 and Cyclin B1) were considerably enhanced, while G1 phase related proteins (CDK6 and Cyclin D1) were suppressed (Fig. 3D). To further strengthen these findings, we also overexpressed HOTAIR transcript in another low HOTAIR-expressed PTC cell line (K1). Again, we are able to observe that cell proliferation and colony formation were significantly enhanced, and cell cycle was shifted toward G2/M phase after HOTAIR overexpression (Additional file 1: Figure S2). Whereas the sub G1 phase was also significantly increased in the HOTAIR-overexpressed K1 cells. Considering the sub G1 phase generally represented the apoptotic cells [19], this phenomenon might be due to the insufficient nutrients to meet the demands during rapid cell proliferation [20].
Numerous studies have shown PTEN/p-AKT pathway heavily involved in regulating all phases of the cell cycle including G1/S [21] and G2/M transitions [22]. PTEN has also been shown to be epigenetically suppressed by HOTAIR [23]. Therefore, we blotted the PTEN and p-AKT in our HOTAIR knockdown and overexpression cellular system. Interestingly, PTEN/p-AKT pathway presented substantially corresponding changes after gain or loss of HOTAIR in PTC cell lines. HOTAIR knockdown in MDA-T32 enhanced PTEN production with decrease of p-AKT/AKT ratio, whereas HOTAIR overexpression in MDA-T41 suppressed PTEN amounts and remarkably increased the p-AKT/AKT ratio (Fig. 3E). These results indicate HOTAIR as a lncRNA can alter PTC cell cycle phases likely through regulating PTEN/p-AKT pathway. DLX1 was epigenetically suppressed by HOTAIR, and lower DLX1 levels in PTC tissues were associated with disease progression HOTAIR mediated recruitment of PRC2 complex, containing enhancer of zeste homolog 2 (EZH2) as a histone-lysine N-methyltransferase enzyme can increase H3K27 trimethylation to silence downstream genes in trans [17]. However, this mechanism has not been explored in papillary thyroid cancer till now. Therefore, we used the TCGA transcriptomic data of PTC tissues to select top 5 genes that were negatively correlated to HOTAIR expression (Additional file 1: Table S1). Then, we run real-time qPCR of these 5 genes in our HOTAIR knockdown and overexpression PTC cellular system. Notably, DLX1 expression showed bidirectional corresponding changes after gain or loss of HOTAIR in PTC cell lines (Additional file 1: Figure S3), suggesting DLX1 might be epigenetically suppressed by HOTAIR. Then, we plotted the expression of DLX1 and HOTAIR in 513 PTC tissues, showing DLX1 is significantly negatively correlated to HOTAIR (r = −0.2872, p < 0.001) (Fig. 4A). Besides, we also observed the highest HOTAIR-expressed PTC cell line (MDA-T32) expressed the lowest DLX1 level (Fig. 4B) and the endogenous DLX1 expression was negatively correlated with HOTAIR levels among four PTC cell lines (Fig. 4C). To further confirm DLX1 as a HOTAIR epigenetically suppressed gene, we blotted the DLX1 protein in our HOTAIR knockdown and overexpression cellular system of PTC cell lines. In line with the results of real-time qPCR, DLX1 proteins were significantly increased in MDA-T32 si-HOTAIR cells and steadily suppressed in MDA-T41 HOTAIR-OE cells (Fig. 4D). Then, we performed chromatin immunoprecipitations on MDA-T32 scramble and si-HOTAIR cells and our results demonstrated the occupancies of EZH2 and H3K27me3 on DLX1 promoter were meaningfully suppressed (p < 0.01 for EZH2 and p = 0.05 for H3K27me3) after HOTAIR knockdown (Fig. 4E). To examine the biological importance of DLX1 in PTC, DLX1 expression between PTC tissues and matched adjacent normal thyroid tissues (n = 59) was compared and its association with disease prognosis was analyzed using TCGA database. Markedly, PTC tissues expressed significantly decreased DLX1 levels (Fig. 5A) and the lower DLX1 expression was associated with poor progression free survival in PTC (Fig. 5B). To prove this association in vivo, we performed DLX1 IHC staining among normal thyroid tissue and different stages of PTC tissues. Indeed, IHC staining shows a significantly stepwise decrease of DLX1 protein from normal thyroid tissues to stage III PTC tissues ( Fig. 5C and D). Overall, our data disclosed DLX1 was epigenetically repressed by HOTAIR and loss of DLX1 associates with advanced PTC stages. DLX1 will likely be a novel marker to evaluate the disease prognosis of PTC.

Discussion
Although patients with PTC generally have good overall survival rate under standard therapy, but there is still a concern for the high disease recurrence, which could reach up to 20% during the long-term follow-up [4]. Also, there is still a difficulty to determine the optimal initial operational choice and postoperative therapeutic strategies (such as if radioiodine ablation is required post total thyroidectomy) for PTC patients. For examples, patients with 1-4 cm intrathyroidal PTC without evidence of contralateral lobe or cervical lymph nodes metastasis can receive either solely thyroid lobectomy or total thyroidectomy with/without radioiodine therapy [24]. One important issue is that the heterogenicity within the histological features and genetic/ epigenetic signatures of PTC have not been thoroughly understood. Therefore, there is a vital importance to Statistical significance was assessed using paired or independent Student T-test; Mann-Whitney U test to examine the differential %Input between two cells, *p < 0.05; **p < 0.01; ***p < 0.001 recognize or discover novel prognosis-associated markers of PTC patients for clinical evaluation.
Recently, lncRNA HOTAIR has been recognized as a practicable biomarker in patients with PTC. The HOTAIR expression in serum or in thyroid tissues collected via fine-needle aspiration biopsies can serve as one of useful markers to differentiate benign nodules and thyroid cancers [10,25]. HOTAIR expression was shown to be significantly increased in PTC tissues, positively correlated with the advanced PTC stages, and negatively associated with overall survival probability of PTC patients [12].
In vitro experiments using PTC cell lines further demonstrate HOTAIR could promote cell proliferation, migration, and invasion via competitively sponging numerous microRNAs including miR-1 [13], miR-488-5p [14], miR-17-5p [15] and miR-761 [16]. In the present study, we observed similar findings that HOTAIR was highly expressed in PTC tissues and its higher expression associated with lower overall survival. Notably, we further found there are differential HOTAIR expression among four PTC cell lines (BCPAP, K1, MDA-T41 and MDA-T32), supporting there is indeed heterogenicity within the category of PTC. We further modulated HOTAIR expression in two newly developed and well-characterized PTC cell lines. We performed HOTAIR knockdown in high HOTAIR-expressed MDA-T32 and conducted HOTAIR overexpression in MDA-T41, which presented significantly lower HOTAIR levels compared to MDA-T32. In line with previous reports, we demonstrated HOTAIR can foster cell proliferation, colony formation and migration, redistribute cell cycle phases toward S phase (DNA replication) and G2/M phase (cell mitosis) and correspondingly change the signaling of PTEN/p-AKT pathway. Overall, HOTAIR is thought as an important epigenetic regulator that vigorously participates in carcinogenesis, tumor growth, progression, and metastasis of PTC. Currently, there is still limited understanding regarding the upstream regulatory mechanisms for HOTAIR transcription. HOTAIR can be upregulated by estradiol in vitro via enriching the bindings of estrogen receptors (ERs) to its promoter [26] and genetic variants such as single nucleotide polymorphisms (SNPs) can also potentially influence the HOTAIR expression with tissuespecific pattern. Indeed, Zhu et al. revealed HOTAIR rs920778 T allele as a functional genetic variant associated with higher HOTAIR levels in both normal thyroid and PTC tissues [27]. HOTAIR expression could also be induced by the tumor microenvironment such as hypoxia [28]. Therefore, the intrinsic factor (genetic variation) and extrinsic factors (estradiol and hypoxia) might coordinate together to modulate HOTAIR expression and contribute PTC tumorigenesis and will require future investigation to clarify in depth.
To extend HOTAIR mediated epigenetic alterations in PTC beyond microRNAs sponge, we examine several genes presented negative correlation with HOTAIR expression in PTC tissues. Then, our attention was centred on the DLX1 gene since it retained negative corresponding changes after gain or loss of HOTAIR in our PTC cellular system. We further identified DLX1 gene was epigenetically repressed by HOTAIR via recruitments of PRC2 (EZH2) and H3K27me3 on its promoter region. Moreover, using the TCGA dataset and IHC staining of PTC tissues, we disclosed that DLX1 expression was significantly reduced in PTC tissues and decrease of DLX1 was associated with advanced PTC stages and lowered progression free survival. These results indicate DLX1 as a novel marker with a promising application for predicting the disease prognosis of PTC patients.
DLX1 belongs to the DLX homeobox family genes that encode transcription factors possessing critical roles in the morphogenesis of craniofacial structures, pharyngeal arches, forebrain, and sensory organs [29]. Particularly, pharyngeal arches are closely linked to the development of thyroid gland. The thyroid originates from a diverticular outgrowth of the primitive pharynx between the first and second pharyngeal arches [30] and the developmental genes such as DLX and HOX genes are both important for patterning the anterior/posterior and dorsal/ventral axes of the pharyngeal arches [31]. HOX genes are the most well-known subset of homeobox genes containing four chromosome clusters (HOXA, HOXB, HOXC and HOXD), which involved in the formation of various body structures during embryonic development [32]. HOTAIR is transcribed from the antisense strand of HOXC cluster in chromosome 12 and has been demonstrated to repress the expression of HOXD genes in chromosome 2 [17]. In this study, we further identified DLX1 gene was epigenetically supressed by HOTAIR. Intriguingly, DLX1 gene just locates next to the HOXD gene cluster, indicating the HOTAIR mediated histone modifications can inhibit several genes in the near position of the same chromosome. Based on RNA sequencing data of GTEx (Genotype-Tissue Expression) Analysis Release V8 (dbGaP Accession phs000424.v8.p2), thyroid tissue is listed as the 2nd rank tissue with high DLX1 expression (only below to the brain tissues), whereas HOTAIR was lowly expressed in thyroid tissue (only above to the whole blood). Therefore, maintenance of low HOTAIR expression and high DLX1 levels in the thyroid gland is essential for its normal development and functionality. In this paper, we further demonstrated their dysregulation can promote the carcinogenesis and disease progression of PTC. However, future investigation is still warranted to ascertain the functional roles of DLX1 in PTC.
In summary, HOTAIR is an important epigenetic regulator for steering the tumorigenesis and disease progression of PTC through modulating the cellular malignancy. We validated HOTAIR can modulate histone modifications to inhibit DLX1 expression and loss of DLX1 was associated with advanced PTC stages and decreased progression free survival. Our results broaden HOTAIR mediated epigenetic regulatory networks in PTC to include histone modifications. Moreover, DLX1 was recognized as a novel prognosis associated biomarker in PTC likely to be practicable for clinical utilization.

Analyze gene expression in PTC tissues and its association with clinical prognosis
The Cancer Genome Atlas (TCGA) thyroid cancer data were obtained from UCSC Xena (http:// xena. ucsc. edu). A total of 59 PTC tissues and matched adjacent normal thyroid tissues were applied to compare the HOTAIR and DLX1 expression. In survival analysis, HOTAIR and DLX1 expression were obtained from 513 and 515 PTC tissues, respectively, and stratified as high or low based on the expression level with significant differences in the survival outcomes and the lowest log-rank P-value among subgroups using Cutoff Finder [33]. HOTAIR expression in 308 TCGA PTC tissues with different genetic alternations including BRAF V600E , NRAS, HRAS mutations and RET fusion were further compared. We also utilized the TCGA data to select the putative HOTAIR suppressed genes that present a significantly negative correlation with HOTAIR expression. The correlation between HOTAIR and DLX1 expression (n = 513) was further plotted and analyzed by Pearson correlation.

HOTAIR knockdown using small interfering (si)RNA transfection
The HOTAIR siRNAs (Cat#N-187951-02, GE Healthcare Dharmacon) and scramble siRNA (Cat#D-001210-01, GE Healthcare Dharmacon) were used to generate the HOTAIR knockdown cells (MDA-T32 si-HOTAIR) and scramble control cells (MDA-T32 Scramble). 10 nM siR-NAs with transfection reagent were added to the cells following the protocols supplied by the manufacturer. Further assays or experiments were performed at 48 h post-transfection.

Generation of constitutive HOTAIR overexpression
The sequence of HOTAIR (NR_003716) was cloned from the LZRS-HOTAIR plasmid (Addgene plasmid #26110) and inserted into the pEGFP-Lv105 vector (Capital Biosciences) as the working HOTAIR plasmid. pEGFP-Lv105 empty vector was used as a control. Then, we applied lentiviral packaging kit (Cat#3D5F03, Origene) in HEK293T cells to produce lentiviral particles. The MDA-T41 cells were transduced with HOTAIR plasmid-or empty vector-lentiviral particles in 8 µg/ml hexadimethrine bromide (Sigma)-containing growth medium to generate MDA-T41 HOTAIR-OE cells or Control cells, respectively. Then, cells were revived in normal growth medium for an additional 24-28 h with subsequent selection in puromycin-containing growth medium.

Cell proliferation assay
The Cell Counting Kit-8 (CCK-8) assay purchased from Dojindo Laboratories (Cat#TJ557, Kumamoto) was used to evaluate cell proliferation rate. All the experimental protocols were performed as described in the manufacturer's instructions. Briefly, 1 × 10 3 cells per well were seeded into a 96-well plate and cultured at 37 °C. At the indicated time points, CCK-8 solution was added to each well with 1:9 ratio (CCK-8: media) and cells were incubated at 37 °C for a further 3 h. The absorbance at 450 nm was measured with a microplate reader (The Synergy ™ HT, Bio-Tek, Taiwan).

Colony-forming unit assay
1 × 10 3 cells per well were seeded in a 6-well plate for 7-14 days at 37 ℃ in 5% CO2. The cells were fixed with 4% formalin for 30 min, washed by PBS and stained with 0.1% crystal violet (Cat#MKCH6258, Sigma). The colony forming units were photographed and counted using Image J.
Wound-healing assay 5 × 10 3 cells per well were seeded in a 12-well plate to reach 80-90% confluence. Then, a small wound was created in each well with a gentle wash to remove cell fragments. Cells were cultured at 37 ℃ in 5% CO 2 and images were taken under a microscope at 0 and 16 h.

Flow cytometric analysis of the cell cycle
Briefly, cells with the culture medium were harvested, washed with PBS following 1300 rpm spinning for 5 min at 4 ℃ × 3 times, fixed with ice cold ethanol (75%), washed with 1% FBS contained PBS following 1300 rpm spinning for 5 min at 4 ℃ twice, then stained with propidium iodide (PI) solution containing 50 μg/ ml PI (Sigma) and 10 μg/ml RNase A (Thermo Fisher Science). Images of the cell cycle were obtained by the FACSCalibur system (BD Biosciences).

Gene expression analysis
Total RNA was extracted from cells using TRIzol reagent and cDNA synthesis was performed using a high-capacity cDNA reverse transcription kit (Cat#00984365, Thermo Fisher Science). SensiFAST SYBR No-ROX mix (Cat#98005, Bioline) was used for running quantitative real-time PCR in a LightCycler 480 (Roche). The ΔCT values of target genes were normalized to the ΔCt of stably expressed reference transcripts (GAPDH). The primer sequences used are listed in Additional file 1: Table S2.

Chromatin Immunoprecipitation (ChIP)
We used the EZ-Magna ChIP ™ A/G Chromatin Immunoprecipitation kit (Cat#17-10086, Millipore) to perform ChIP experiments as the standard protocols. Briefly, in vivo cross linking was conducted via adding 1% formaldehyde to the cells (1 × 10 6 per ChIP) for 10 min. Sonication (2*10 bursts of 30 s ON/OFF at high-level output in the sonicator (XL2015, Misonix)) of the cross-linked chromatin was performed to generate < 500 bp DNA fragments with confirmation via running electrophoresis on the agarose gel stained with DNA VIEW (Cat#TT-DNA01, TOOLS). 1% of chromatin supernatant was collected as the input. Then, antibodies with ChIPAb + H3K27me3 (Cat#17-622, Millipore), ChIPAb + EZH2 (Cat#17-662, Millipore) or mouse IgG (Cat#12-371B, Millipore) were used to perform immunoprecipitation. For binding the antibody/antigen/DNA complex, magnetic protein A/G Beads (Cat# CS204457, Millipore) were applied. Then, ChIP samples were going through washing, reversal of cross-linking, and ChIP DNA isolation. The eluted ChIP DNA was used as the template for real-time qPCR using SYBR Green. To assess the occupancy of H3K27me3 and EZH2 on the DLX1 gene, the primers target on DLX1 promoter region were designed as listed in Additional file 1: Table S2.

Immunohistochemistry staining of normal thyroid and papillary thyroid cancer tissues
The paraffin embedded sections of normal thyroid tissues (n = 22) and papillary thyroid cancer tissues with different stages (n = 52 with stage I, n = 20 with stage II, n = 15 with stage III and n = 3 with stage IVA) were purchased from US Biomax, Inc (Cat#TH8010a, Cat#TH208, Cat#TH961). Immunohistochemistry (IHC) staining of DLX1 was performed as the standard protocol including deparaffinized, rehydrating, antigen retrieval, immunohistochemical staining using the antibody against DLX1 (Cat#PA5-28899, Thermo Fisher Science), dehydrating and stabilizing with mounting medium and viewing the staining under the microscope. The staining intensity from 0 to 3 was scored, with 3 referred as the section with maximum intensity, while 0 indicated negative. The percentage of staining was estimated, and 100% staining was scored as 1. The IHC scores were calculated by staining intensity*staining percentage and assessed by an independent pathologist.

Statistical analysis
Graphpad Prism 6.0 and SPSS version 22 were used for data analysis. The details of statistical methods were described in the figure legends.
Additional file 1: Figure S1. Comparison of HOTAIR expression among PTCs with different genetic background. Figure S2. Overexpression of HOTAIR in the low HOTAIR-expressed K1 cell enhanced proliferation, colony formation and shifted the cell cycle toward G2/M phase. Figure S3. DLX1 expression presented corresponding changes after HOTAIR modulation and had an endogenously negative correlation with HOTAIR levels in PTC cell lines. Table S1. Top 5 genes presented significantly negative correlation with HOTAIR expression in PTC tissues of TCGA data. Table S2. Used primers sequences. Figure S4. The corresponding changes of protein levels after HOTAIR modulation in MDA-T32 and MDA-T41 cell lines. (A) Protein markers of different cell phases, PTEN and p-AKT/AKT ratio were blotted on Scramble/si-HOTAIR cells (B) Protein markers of different cell phases, PTEN and p-AKT/AKT ratio were blotted on Control/HOTAIR-OE cells (C) DLX1 were blotted on Scramble/si-HOTAIR and Control/HOTAIR-OE cells.